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  • UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)

    UvA-DARE (Digital Academic Repository)

    Familial hypercholesterolemia: the Dutch approach

    Huijgen, R.

    Link to publication

    Citation for published version (APA):Huijgen, R. (2012). Familial hypercholesterolemia: the Dutch approach

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    Download date: 08 Feb 2018

    http://dare.uva.nl/personal/pure/en/publications/familial-hypercholesterolemia-the-dutch-approach(215da265-f714-4dbd-8e1a-9c1b7b9e4cde).html

  • Chapter 6

    Cardiovascular Risk in Relation to

    Functionality of Sequence Variants in

    the Gene Coding for the Low-Density

    Lipoprotein Receptor

    A study among 29,365 individuals tested for 64 specific LDLR sequence variants

    Roeland Huijgen, Iris Kindt, Joep C. Defesche and John J.P. Kastelein

    European Heart Journal 2012;33(18):2325-2330.

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    Chapter 6

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    AbSTRACT

    Background: A plethora of mutations in the LDL-receptor gene (LDLR) underlie the clinical phenotype of familial hypercholesterolemia (FH). For the diagnosis of FH, it is important, however, to discriminate between pathogenic and non-pathogenic mutations. The aim of the current study was to assess whether true pathogenic mutations were indeed associated with the occurrence of coronary artery disease (CAD) when compared to non-functional variants. The latter variants should not exhibit such an association with CAD.

    Methods: We assessed 29,365 individuals tested for the 64 most prevalent LDLR variants. First, we determined pathogenicity for each of these sequence variants. Subsequently, a Cox-proportional hazard model was used to compare event-free survival, defined as the period from birth until the first coronary artery disease (CAD) event, between carriers and non-carriers of LDLR mutations.

    Results: Fifty-four sequence variants in the LDLR gene were labeled as pathogenic and 10 as non-pathogenic. The 9,912 carriers of a pathogenic LDLR mutation had a shorter event-free survival than the 18,393 relatives who did not carry that mutation; hazard ratio 3.64 (95%CI: 3.24 to 4.08; p

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    Cardiovascular risk and functionality criteria for the LDLR

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    InTRODuCTIOn

    Familial hypercholesterolemia (FH) is a frequent autosomal co-dominant disorder of lipoprotein metabolism, with 1 in 500 persons affected with heterozygous FH in most Western countries.1 Patients with FH have elevated plasma low-density lipoprotein-cholesterol (LDL-C) levels and an increased risk of premature coronary artery disease (CAD).1, 2 Defects in genes that code for proteins involved in hepatic clearance of LDL-C underlie this hereditary disorder.1 In fact, more than a 1000 different mutations in the genes coding for the LDL-receptor (LDLR), apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9) are now known to cause FH.3

    The identification of a mutation that underlies FH in a particular family enables genetic testing of family members for the presence of the same mutation and makes it possible to initiate effective medical management before the cardiovascular consequences of FH become clinically manifest. This notion has led to the implementation of a nationwide genetic cascade screening program for FH in the Netherlands and approximately 27,000 subjects with FH have been found and treated since 1994.4

    However, as widely appreciated, not every sequence variant in LDLR, APOB or PCSK9 results in an FH phenotype. If a novel sequence variant in one of those genes is identified in a patient with a clear clinical FH diagnosis, one has to rely on in silico or in vitro studies to ascertain pathogenicity. However, in vitro studies are cumbersome and therefore rarely performed. Also, the in silico results do not always correspond with the clinical observations.5 An alternative strategy is to perform cascade screening for such a novel variant and to identify new carriers of that specific variant to determine whether these carriers express an FH phenotype. Accordingly, we recently validated specific criteria that can discriminate pathogenic from non-pathogenic mutations, using lipoprotein and lipid levels as well as the use of lipid-lowering medication.5 Since then, collected a much larger cohort of carriers of non-pathogenic variants in order to assess possible consequences of these variants on occurrence of cardiovascular disease.

    In the present study, we compared cardiovascular risk between individuals with established pathogenic FH mutations and those with sequence variants in LDLR that we consider non-pathogenic. Here, we present our results.

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    Chapter 6

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    METHODS

    In this observational study we used a stepwise approach. First, we applied our criteria to assess functionality of all variants we have identified in LDLR in order to expand the number of individuals with pathogenic and non-pathogenic variants. Second, we compared CAD risk between carriers and non-carriers of all non-pathogenic variants and, similarly, of all pathogenic mutations.

    Study populationThe functionality criteria and event free survival were assessed in individuals who were screened by genetic testing in the period between January 1994 and December 2010. Of these subjects, lipoprotein profiles, an extensive history of cardiovascular disease, medication use, and specific carrier status were collected at the time of molecular diagnosis. We excluded index patients from the current analysis to avoid clinical sampling bias. The cascade screening program was launched by the Ministry of Health of the Dutch Government and approved by the National Ethics Committee. All participants gave written informed consent for genetic analysis.

    Criteria to test functionalityThe three criteria to establish functionality of a specific DNA variant have been described before 5 and were: (1) a mean LDL-C level above the 75th percentile for age and gender in untreated individuals carrying such a mutation, (2) statistically significantly higher mean LDL-C levels in untreated carriers compared to untreated non-carriers, and (3) a statistically significant higher percentage of medication users among carriers compared to non-carriers at the time of molecular screening. We considered a mutation to be non-pathogenic when none of the three criteria were met.

    All genetic variants had to be tested in at least 50 untreated subjects per variant. Additionally, the in silico analyses were applied to all prevalent variants by using the Alamut software (version 2, Interactive Biosoftware, Rouen, France: SIFT and PolyPhen prediction). Mutations were described according to the nomenclature as proposed by den Dunnen and Antonarakis,6 and we used the description based on the effect of the mutation on the protein.

    Coronary artery diseaseCAD was defined as a history of one of the following non-fatal cardiac outcomes: a) sudden cardiac arrest; b) myocardial infarction; c) coronary artery bypass graft, or d)

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    Cardiovascular risk and functionality criteria for the LDLR

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    percutaneous transluminal coronary angioplasty. CAD event-free survival was defined as the period from year of birth until the year of the first CAD event or censoring at the moment of genetic screening.

    Statistical analysisDifferences in LDL-C levels between subgroups were compared by means of the unpaired Student t-test. Proportions of subjects using lipid-lowering medication were compared with the chi-square test. We compared survival of mutation carriers with that of family members without mutations using Kaplan-Meier survival analysis. A Cox-proportional hazard model was used to comp